Triazinoindole compounds

ABSTRACT

3-CARBOXYALKYLAMINOTRIAZINO(5,6-B)INDOLES ARE PREPARED MICROBIOLOGICALLY BY ACTION OF A VARIETY OF ORGANISMS, PREFERABLY NOCARDIA RUBRA ATCC 19557, ON THE CORRESPONDING 3-HYDROXYALKYLAMINOTRIAZINOINDOLE. THE PRODUCTS HAVE ANTIVIRAL ACTIVITY, PARTICULARLY AGAINST RHINOVIRUSES.

United States Patent W Int. Cl. C12d 1/02 U.S. Cl. 195-51 3 ClaimsABSTRACT OF THE DISCLOSURE 3 carboxyalkylaminotriazino[5,6 b]indoles areprepared microbiologically by action of a variety of organisms,preferably Nocardia rubra ATCC 19557, on the corresponding 3hydroxyalkylaminotriazinoindole. The products have antiviral activity,particularly against rhinoviruses.

This is a division of application Ser. No. 584,657 filed Oct. 6, 1966,now US. Pat. 3,453,276.

The present invention relates to triazinoindole compounds havingantiviral activity. In particular, the invention relates toas-triazino[5,6-b]indoles substituted at the 3-position With acarboxyalkylamino grouping.

The compounds of the invention are represented by the followingstructural formula:

\N Jaanauztpnoo01st wherein:

R is lower alkyl of l-4 carbon atoms or phenylalkyl of 7-9 carbon atoms;

R is hydrogen, methoxy, chloro, or nitro; and

n is a positive integer from 2 to 7.

A preferred group of compounds is represented by Formula I when'R ishydrogen and R is methyl.

The present invention also includes the pharmaceutically acceptable acidaddition salts of the compounds of Formula I. Among these salts are thehydrochloride, hydrobromide, sulfate, and maleate, all of which areprepared by well-known salt-forming techniques. The invention alsoincludes the alkali metal, ammonium, and simple amine salts of theinventive compounds. These latter salts constitute the form in which thecompounds exist when in basic solution and are formed by conventionalbasification and isolation techniques. Since the compounds of Formula Ipossess both amine and carboxylic acid functions, they may also exist ina zwitterionic form in which one of the nitrogen atoms bears a positivecharge and the carboxylic acid function exists as its carboxylate anion.The invention is intended to-comprehend the compounds as represented byFormula I or by the dipolar zwitterionic species, as well as in the formof their acid and basic salts and hydrates.

The compounds of Formula I are prepared either microbiologically orchemically. When a microbiological 3,595,752 Patented July 27, 1971method of preparation is utilized, the starting materials are theprimary alcohols corresponding to the product carboxylic acids. Thesealcohols are represented by Formula II, where R, R and n are as definedin Formula I.

These compounds are described in Belgian Pat. 653,089 or are prepared bymethods described therein. The preferred method of preparing thesestarting materials is by condensing an appropriately substituted3-mercaptotriazinoindole with a hydroxyalkylamine.

Many species of microorganisms convert an alcohol of Formula II to acarboxylic acid of Formula I. Among these are many representativespecies of fungi, bacteria and actinomycetes, including members of thegenera Scopulariopsis, Nocardia, Streptomyces, Geotrichum, Pseudomonas,Penicillium, Beauvaria, Aspergillus, Pachybasium, Cunninghamella,Protaminobacter, Corynebacterium, Arthrobacter, Bacillus, and manyothers. The organism of choice is Nocardia rubra (ATCC 19557), since theabove product is formed in higher yield with few (if any) competing sidereactions. However, the process is not limited to this species.

These microorganisms are cultivated in liquid media, such as nutrientbroth, yeast extract broth, Trypticase Soy Broth, soybean meal broth, orpeanut meal broth. The media should contain sources of available carbon,nitrogen, and minerals. In our experiments, best results were obtainedusing the glucose-amm-onia-soybean medium shown in Example 1.Carbohydrates, such as starches, dextrins, and sugars, including hexosesand pentoses, may be used to furnish the energy and carbon requirementsof the microorganisms. Other sources of carbon may also be used, forexample, citric acid and its salts, sodium acetate, alcohols or fattyacids. Sources of nitrogen in assimilable form can be made available bysoluble or insoluble vegetable or animal protein and protein derivativessuch as soybean meal, casein, meat extracts, corn steep liquor, yeastextract and peptones. Amino acids or ammonium salts can also be used.Minerals naturally present in the above carbon and nitrogen sources areusually sufiicient to satisfy the requirements of the microorganisms. Asterile air supply must be maintained during the fermentation. This canbe accomplished by exposing a large surface of the growth medium to theatmosphere with constant agitation, or alternatively by the use ofsubmerged aeration devices. Aeration at a rate of about 0.5 to 2.0volumes of air per volume of growth medium per minute producessatisfactory results.

During the fermentation the temperature should be maintained within arange of about 23 C. to 32 C., preferably from about 25 C. to 30 C.

Optimum growth of the microorganisms is achieved when the pH of thefermentation is maintained Within a range of pH 6.0 to 6.8. This may beaccomplished by the intermittent addition of mineral acids or bases toadjust the pH or also by the incorporation of buffering agents in thefermentation medium. Buffering agents such as calcium carbonate orpotassium dihydrogen phosphate may be used. In the case of N. rubra thetransformation appears to take place most rapidly after maximum growthhas terminated, the vegetative hyphae have fragmented, and the reactionof the medium is maintained in the range pH 7 .5- 8.0.

The substrate alcohol may be added to the culture as a finely dividedsolid or in solution in a suitable solvent, such as ethanol or methanol.We prefer to convert the substrate compound to its hydrochloride form,which is more readily soluble in the lower alcohols and water. Theaddition of the substrate to the microbial culture should he made underaseptic conditions. The incubation and aeration of the culture iscontinued in order to bring about the transformation of the substrate.Alternatively the substrate may be added to the fermentation medium atthe time the medium is first inoculated with the culture ofmicroorganism.

The fermentation or biotransformation process is continued until themaximum amount of product has accumulated. This usually occurs within atime period of about 4-48 hours, and is most easily determined byperiodic analysis of the fermentation system. This analysis can best becarried out chromatographically, as this method gives a quick andaccurate representation of the types and relative concentrations of thecompounds present. We have used both paper and thin layer chromatographyfor analysis. The actual methods used are cited in the examples below.We have found that product yields can be increased by continued feedingof additional supplies of substrate to the fragmented culture, providedthat the transformation culture is also supplemented with glucose andthe reaction is controlled at pH 7.58.0.

When the transformation has progressed to its maximum stage (as shown bychromatographic analysis), the fermentation is terminated and thecompounds, both untransformed substrate and the transformation products,are recovered. This is most commonly done by extraction of the aqueousfermentation broth with organic solvents which are immiscible withwater, n-butanol, or other immiscible solvents of like polarity, aremost satisfactorily used to extract the product carboxylic acid.Alternatively the carboxylic acid can be converted directly to itsmethyl or other lower alkyl ester by addition of the appropriate alkanolto an acidic mixture of the carboxylic acid, and the ester is thenreadily extracted from the aqueous broth with chloroform. The wholefermentation broth, including microbial cells and aqueous supernatantfluid, can be extracted, or alternatively the cellular mass of themicroorganism can first be separated from the aqueous supernatant fluidby centrifugation or filtration. In the latter case extraction ofcompounds from the microbial cellular mass is best accomplished by theuse of methanol or other residual traces of water are removed withsuitable drying agents, such as anhydrous sodium sulfate. The driedsolvent extract is then concentrated in vacuo to dryness at temperaturesgenerally not exceeding 60 C. A brownish- 'colored residue results whichcontains the compounds of interest as well as many solvent extractablemiscellaneous compounds produced as a result of microbial metabolism. Itis necessary to remove these contaminating materials in order to obtainthe product in a purified state.

In many cases the product is present in high enough concentration thatit can be readily crystallized from a methanol or other lower alkanolsolution of this dried residue after preliminary decolorization withactivated charcoal. In this case, however, the crystalline precipitatemay contain a mixture of the free acid and its lower alkyl ester. If theester has been extracted, the residue containing ester is redissolved ina small volume of hot CHCl and precipitated with a large excess ofhexane. The ester is then recrystallized from CHCl -MeOH. The ester isreadily hydrolyzed by aqueous acid at 120 for a few minutes, and thefree acid then crystallized from appropriate solvents. The lower alkylesters, being readily hydrolyzed -to the corresponding carboxylic acids,are useful for the preparation of the acids and are therefore part ofthe present invention.

If a mixture of products results from the fermentation process, or if asignificant amount of untransformed substrate remains, more elaboratepurification procedures are required. We have used column chromatographyfor these purifications. The methods used consist in general of thegradient elution of the products from a column of adsorbent material(such as silica or cellulose) by mixtures of judiciously chosen organicsolvents. The presence of the separated compounds in the solventfractions obtained after column chromatography is most easily determinedby paper or tin layer chromatographic analysis of aliquot samples. Theappropriate fractions containing purified compounds are-pooled,concentrated in vacuo, and the purified compounds are finallycrystallized from appropriate solvent mixtures. The following exampleillustrates a typical experiment for the microbial transformation of3-(3-hydroxypropylamino)-5-methyl-as-triazino[5,6 b] indole to3-(2-carboxyethylamino)-5-methyl-as triazino [5,6-b]indole, therecovery, isolation and purification of the product.

' EXAMPLE 1 3-(2-carboxyethylamino)-5-methyl-as-triazino [5,6-b]

' indole Nutrient agar slants of Nocardia rubra ATCC 19557 were used toprepare 50 ml. broth cultures in a sterile medium of the followingcomposition:

Medium SBM-6 G./liter 1.0% Soybean meal (Archer-Daniels-Midland) 10 1.0Cerelose 10 1.0 (NH SO 10 0.01 M so n o 0.1 0.01 KH PO 0.1 0.05 NaCl 0.5

Tap water, q.s., 1000 ml. pH 6.0-6.5 without adjustment, then add 0.5%

CaCO 5 The flasks were incubated at 25 with continual shaking on arotary shaker describing a 2-inch circle at 200 rpm. After 24-48 hoursincubation, just before microbial growth had reached its maximum, theabove cultures were used to inoculate larger volume of the same medium,using at least 10% (v./v.) inoculu-m. This scale-up of the microbialculture was continued until a SO-liter culture was obtained.

The 50-liter culture was used for the actual transformation of substrateto product. This was carried out in a l30-liter Fermacell batchfermentator (New Brunswick Scientific Co.) under the followingconditions:

Aeration: 1.8 ,c.f.m. (1.0 v.v.m.); Head pressure: 10

p.s.i.g. Agitation: 300 r.p.m.;Temperature: 30

Foaming was controlled by the intermittent addition of sterile UCON oilLB625 (Union Carbide Company) as required.

After '24 hours mycelial growth had reached its maximum state, thevegetative hyphae had begun to fragment, and the microorganism had begunto synthesize a light orange pigment (which is characteristic of thisstrain N. rubra).

50 g. of 3-(3-hydroxypropylamino)-5-methyl-as-triazino[5,6-b]indo1e (asthe hydrochloride in 200 m1. ethanol) was added aseptically, togetherwith an additional 500 g. of sterile glucose in solution, aeration wasincreased to 3.6 c.f.-m. (2.0 v.v.m.), agitation was increased to 400r.p.m. and the reaction (which had dropped to pH 6.0) was raised to andmaintained at pH 7.6 with NaOH. The

progress of the transformation was followed by chromatographic analysis,as follows:

Samples were taken from the fermentor aseptically, and 1.4 ml. aliquotportions were extracted with 0.5 ml. n-butanol. The butanol extractswere spotted on Whatman No. 1 paper and developed (ascending) in amixture of npropanol; conc. NH OH: water (6:321 or 8: 1:1 v./v./v.). Thedried chromatograms were examined under ultraviolet light. The substrateand product appeared as yellow fluorescent spots.

Chromatographic analysis showed that all of the substrate wasmetabolised within 8 hr., and the carboxyethylamino compound was theonly product detectable under these conditions.

The transformation was terminated by chilling the culture to thereaction was adjusted to pH 2 with HCl, and the cells were separatedfrom the broth by centrifugation on a Sharples Supercentrifuge. The massof cells showed only traces of product, so were discarded. The aqueousbroth supernatant solution, containing high concentrations of thedesired product, was adjusted to pH 3.5-4.0 carefully with NH OH, and aprecipitate of yellow crystals of crude product was collected. Theremaining broth was extracted three times with n-butanol. These extractswere pooled and concentrated to dryness in vacuo at a temperature lessthan 40.

The dried residue was dissolved in methanol, acidified with HCl,filtered, decolorized with activated charcoal, and adjusted to pH3.5-4.0 with NH OH. A fine precipitate of pale yellow needles wasobtained, which proved to be a mixture of the free acid with traces ofits methyl ester.

The pooled batches of crude product acid crystals plus the traces of themethyl ester present were dissolved to make a concentrated solution inaqueous HCl. This was heated at 120 for 15 minutes in the autoclave,cooled, and adjusted to pH 3.5-4.0 with NH OH. The pale yellow needleswhich precipitated were filtered off and washed with water. The yellowfiltrate, containing some of the product as the ammonium salt, wasacidified with acetic acid, and another precipitate of yellow needleswas obtained. The excess acetic acid was removed by washing the crystalswith ether or CHCI in which the product was insoluble. The washedcrystals were recovered by vacuum filtration and dried overnight invacuo at 60, M.P. 247-250 with decomposition. Paper chromatographyshowed a single, yellow fiuorescing spot, characteristic of the acidproduct, at R 0.45 in PrOH :NH OH :H O (8:1:1).

EXAMPLE 2 By utilizing the following starting materials as substratesand carrying out the procedures described in the previous example, thefollowing products are obtained, respectively.

Starting materials 3- 3-hydroxypropylamino -S-chloro-5methyl-as-triazino5,.6-b indole 3- 4-hydroxybutylamino) -5-methyl-as-triazino 5,6-b indole3 3 -hydroxypropylamino) -5-ethyl-as-triazino 5,6-b 1 indole 3- (6-hydroxyhexylamino -5-methyl-a s-triazino 5,6-b indole Products3-(2-carboxyethylamino) -8-chloro-5-methylas-triazino 5,6-b] indole 3-3-carboxypropylamino -5-methyl-as-triazino[5,6-b]indole 3-(Z-carboxyethylamino -5-ethyl-aS-triazino 5,6-b indole 3- 5 -carboxypentylamino -5 -methyl-as-triazino [5,6-b] indole 6 When a chemicalmethod of synthesis of the compounds of Formula I is employed, thestarting materials are the 3-hydroxy-as-triazinoindoles of Formula III,where R and R are as defined in Formula I.

III

These compounds are prepared as described in Belgian Pat. 653,089, bytreating the corresponding mercapto compounds, also described therein,with hydrogen peroxide or chloroacetic acid. The hydroxy compound isthen converted to a 3-chloro compound, preferably by reaction withphosphorus oxychloride and N,N-dimeth ylaniline. The chloro compound isthen condensed with a cyanoalkylamine to give a 3-cyanoalkylaminotriazinoindole, and the cyano group is hydrolyzed by acid, preferably amixture of hydrochloric and acetic acids, to give the product carboxylicacid.

The following examples illustrate the manner of preparing the compoundsof the invention by chemical means, but are not to be considered aslimiting the scope of the compounds preparable thereby.

EXAMPLE 3 3- Z-carboxyethylamino -5'-methyl-as-triazino [5,6-b] indole Amixture of 1.0 g. of 3-hydroxy-S-methyl-as-triazir1o[5,6-b]indole, 4.0ml. of POCI and 2.0 ml. of N,N-dimethylaniline is heated under refluxfor 15 minutes. The dark brown mixture is cooled slightly and pouredonto -100 g. of cracked ice. The crude 3-chloro-5-methyl-as-triazino[5,6-b1indole is then filtered OE andrecrystallized from ethanol; M.P. 219-22l.

A mixture of 10.3 g. of this 3-chloro compound, ml. of3-aminopropionitrile, and ml. of chloroform is heated under reflux on asteam bath for 18-20 hours. The chloroform is removed in vacuo and theresidue is heated with 10 volumes of water. The aqueous mixture isfurther evaporated to remove the last traces of chloroform, whereuponthe solid which precipitates is removed by suction filtration. Theresulting solid 3-(2-cyanoethylamino) 5 methyl-as-triazino[5,6-b]indoleis purified by recrystallization from ethanol to give a light brown toyellow granular product, M.P. 200-2015.

A stirred solution of 2.5 g. of this cyano compound, 25 ml. of 20% HCl,and 25 ml. of glacial acetic acid is heated under reflux for 18 hours.The reaction mixture is then evaporated to dryness on a rotaryevaporator. The residual solid is dissolved in excess 5% NaHCO solution,and the solution is clarified by filtration through a Super-Cel mat. ThepH of the filtrate is adjusted from 8.5 to 4.5 by adding glacial aceticacid and the precipitated fully hydrated Solid is filtered oil? anddried at 60 in a vacuum oven. This compound, M.P. 252253, is aone-eighth hydrated form of the carboxyethylamine product.Recrystallization from dimethyl formamide gives the anhydrous product,M.P. 248-249".

EXAMPLE -4 By starting with the following starting materials, andperforming the chlorination, condensation, and hydrolysis reactions asdescribed in Example 3, the following products are obtained,respectively.

Starting materials 3-hydroxy-S-benzyl-as-triazino 5,6-b] indole3-hydroxy-5-methyl-as-triazino 5,6b] indole3-hydroxy-8-chloro-5-methyl-as-triazino [5,6-b] indole3-hydroxy-8-nitro-S-methyl-a -triaZinO [5,6-b]indole3-hydroxy-8-methoxy-5-propyl-as-triazino [5,6-b] indole Cyanoalkylamine3-aminopropionitrile -aminovaleronitrile 3-aminopropionitrile8-aminooctanonitrile 3-aminopropionitrile Products 3-(2-carboxyethylarnino -5-benzyl-as-triazino [5,6b] indole 3- (4-carboxybutylamino) -5 -methyl-as-triazino [5,6-b]indo1e 3 2-carboxyethylamino -8-chloro-5 methyl-astriazino [5,6-b1indole 3(7-carboxyheptylamino -8-nitro-5-methyl-as-triazino [5 ,6-b] indole 3-(Z-carboxyethylamino) -8-methoxy-5-propyl-as-triazino [5,6-b] indole Thecompounds of this invention are antiviral agents. They are activeparticularly against rhinoviruses. They are formulated for use bymethods well known to the skilled pharmaceutical chemist. They areintended to be administered orally in the form of a tablet or capsule orintranasally as a 05-10% suspension or solution. The tablets andcapsules may include such common pharmaceutical excipients as sodiumcarboxymethyl cellulose, terra alba, sucrose, starch, lactose, andmagnesium stearate. They are intended to be administered in doses of0.5-5 g./ day, preferably 1-2 g./ day.

EXAMPLE 5 3 (2-carb oxyethylamino) -5 methyl-astriazino[5,6-b] indoleThe microbial transformation was carried out essentially as inExample 1. The following procedure was then used to isolate and purifythe product. This procedure has the advantage that it is better suitedto large-scale fermentation recovery processes with fewer losses inproduct yield.

The acidified (HCl, pH 1-2) aqueous broth supernatant fluid wasconcentrated in vacuo at T 50 to its volume. This yielded a dark brownsyrup from which precipitated a brown sludge of salts and other solids.The syrup and sludge were separated by decantation and treatedseparately with MeOH. In the presence of high concentrations of MeOH andHCl, the acid product orginally present Was rapidly converted to itsmethyl ester. The MeOH solutions containing the methyl ester (and manyimpurities) were concentrated in vacuo to a dark brown, oily residue.This was dissolved in a minimal volume of boiling CHCl and then pouredinto a large excess (10-20 volumes) of n-hexane. The methyl ester of theacid product precipitated as an amorphous mustardyellow powder, whichwas filtered ofi. Recrystallization from CHCl -MeOH containing a fewdrops NH OH yielded fine, pale yellow plates, M.P. 189-190", R 0.95 inPrOH: NH OH: H O (8:1:1).

The methyl ester of the desired carboxylic acid product was hydrolyzedby boiling with aqueous HCl at The cooled hydrolysate, a deep yellow, aclear solution, was adjusted to pH 6-7 with NH OH, and some unhydrolyzedmethyl ester crystallized within a few minutes and was filtered off. Theclear yellow solution of the ammonium salt of the carboxylic acid wasreacidified with acetic acid to pH 3.0-3.5, and within a few minutesfine yellow needles precipitated. These were filtered, washed free ofresidual salts with water, and dried overnight in vacuo at 60. Theproduct melted at 247-250 with decomposition.

EXAMPLE 6 The microbial transformation was carried out essentially as inExample 1 except that the substrate was added 1 as a finely crushedpowder suspended in a little water to give a final concentration ofsubstrate of 1 mg./ml. The transformation culture (50 m1.) contained,therefore, a total of 50 mg. substrate.

The transformation was complete within 36 hours, and chromatographicanalysis showed that no substrate or other detectable products werepresent.

The product was isolated essentially as described in Example 5 to give afinal yield of 35.6 mg. crystalline free acid (71.2% overall yield,direct weight basis).

1 To a 50 m1. culture of N. mbm ATCC 19557 in medium SBN-G.

We claim:

1 A process for preparing a compound of the formula comprisingsubjecting a compound of the formula:

where R is lower alkyl of 1-4 carbon atoms or phenylalkyl of 7-9 carbonatoms; R is hydrogen, methoxy, chloro, or nitro; and n is a positiveinterger from 2 to 7; or a pharmaceutically acceptable acid additionsalt or an alkali metal or ammonium salt thereof, to the action ofNocardia rubra ATCC 19557 in an aqueous, aerated nutrient medium.

2. A process as claimed in claim 1, where R is methyl and R is hydrogen.

3. A process as claimed in claim 2, where n is 2.

References Cited UNITED STATES PATENTS 3,505,341 4/1970 Elslager et a1.5l

ALVIN E. TANENHOLTZ, Primary Examiner

